Antisense oligonucleitide containing compositions and method of forming duplexes

ABSTRACT

Replacement of the natural nucleotides with unnatural zwitterionic nucleotides having a cationic moiety tethered to the base (or analog thereof) results in oligodeoxynucleotides with diminished charge but undiminished ability to complex with DNA at low ionic strengths. We have now discovered that DNA can be made fully zwitterionic by introducing tethered cationic moieties to the bases without affecting duplex formation. The resulting oligonucleotides have the further advantages of being nuclease resistant.

This invention was made with Government support under Grant No.GM47375-01, awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

This is a Continuation of application Ser. No. 08/214,603 filed Mar. 18,1994, now U.S. Pat. No. 5,596,091.

The present invention relates to novel antisense oligonucleotidesderived from zwitterionic monomers. These oligonuclectides are capableof forming duplexes with natural DNA which are resistant to nucleasedegradation and are useful in diagnostic and therapeutic applications.

BACKGROUND OF THE INVENTION

Antisense oligonucleotides are synthetic oligonucleotides which aredesigned to bind to RNA by Watson-Crick base paring. This binding, orhybridization can result in the selective inhibition of RNA expression.Additionally, the antisense oligonucleotides may block gene expressionby inhibition of replication or transcription of DNA by Hoogsteenbonding. This type of bonding results in the formation of a triple helixwherein the oligonucleoide analog binds in a sequence-specific manner inthe major groove of the DNA duplex structure. A number of cellularprocesses can be inhibited depending on where the oligonucleotidehybridizes on regions of DNA or mRNA. To be effective as therapeuticagents, oligonucleotides must reach the interior of target cellsunaltered. This requires the oligonucleotides to be able to penetratethe cell membrane and to be resistant to intra- and extracellularnucleases. Additionally, antisense oligonucleotides can be used fordiagnostic purposes by coupling the oligonucleotide to a suitableimaging agent (i.e., radiolabel, fluorescent tag, or biotin) or solidsupport.

Natural oligonucleotides are negatively charged and do not easilypenetrate the cell membrane. Additionally, they are susceptible todegradation by nucleases which cleave the phosphodiester linkage. Forthese reasons, efforts to prepare pharmaceutically activeoligonucleotides have focused on synthetic analogs which address theseproblems.

A number of strategies have been employed in the preparation ofantisense oligonucleotides including, replacement of one non-bridgingoxygen in phosphodiester linkages with sulfur (see, Cohen, et al., U.S.Pat. Nos. 5,264,423 and 5,276,019); replacement of both non-bridgingoxygens in the phosphodiester linkages with sulfur (see, Brill, et al.,J. Am. Chem. Soc. 111:2321 (1989)); use of nonionic alkyl and arylphosphonates in place of the phosphate linking group (see, Miller, etal., U.S. Pat. Nos. 4,469,863 and 4,757,055); and, replacement ofphosphorus atoms with carbon or silicon (see, Stirchak, et al., J. Org.Chem. 52:4202-4206 (1987) and Cormier, et al., Nucleic Acids Res.16:4583 (1988), respectively). These strategies and related efforts arethe subject of a recent review (see, Uhlmann, et al., Chem. Reviews90:543-584 (1990)).

More recently, others have focused on the preparation of charge-neutralantisense oligonucleotides which have zwitterionic moieties attached tothe phosphodiester backbone (see, Cook, WO 93/15742 (1993)).

Base modification has been virtually ignored as a method by which toengineer oligonucleotides for use as antisense agents. However, naturehas employed a design strategy of attaching a positively charged speciesto a thymidine base. Thus, bacteriophage φW-14 is known to replaceapproximately half of the thymines present in its DNA with positivelycharged α-putrescinylthymine. This results in approximately onehypermodified base every eight nucleotides on average. These and otherhypermodified bacteriophage DNAs have been shown to resist a variety ofendonucleases and some exonucleases as well. While it is known fromthese and other examples that the DNA major groove will tolerate somesubstitution without a significant deleterious effect on duplexformation, little information is available about the effect ofintroducing contiguous, bulky, charged modifying groups into the majorgroove.

SUMMARY OF THE INVENTION

We have discovered that modified oligonuclectides can be prepared insubstantially pure form having diminished charge but undiminishedability to complex with natural DNA at low ionic strengths. Thesemodified oligonucleotides are prepared by replacing natural nucleotideswith unnatural zwitterionic nucleotides having a cationic moietytethered to the base. The modified oligonucleotides have the advantagesof being membrane permeable and nuclease resistant, and can further formduplexes even when completely zwitterionic. As a result, the modifiedoligonucleotides of the present invention may be used in variousapplications such as where antisense therapy is desired, they may becoupled with an imaging agent for use in diagnostic applications, orthey may be used in in vitro hybridization assays where nucleaseresistant capture probes or signal probes are desired.

The following abbreviations are used herein: Bz, benzoyl; CPG,controlled pore glass; DCC, dicyclohexylcarbodiimide; DMF,dimethyrformamide; DMSO, dimethylsulfoxide; DMT, dimethoxytrityl; EDTA,ethylenediaminetetraacetic acid; FAB, fast atom bombardment; HPLC, highperformance liquid chromatography; HRMS, high resolution massspectrometry; IR, infrared spectrometry; NMR, nuclear magnetc resonancespectrometry; PAGE, polyacrylamide gel electrophoresis; PEG,polyethylene glycol; TBDMS, tert-butyidimethylsilyl; TEAA,triethylammonium acetate; and TLC, thin layer chromatography.

As used herein the term "natural DNA" refers to oligomers composed onlyof the naturally occuring nucleotides. The oligomers may be eithersingle- or double-stranded.

The term "oligonucleotide" refers to a single or double-stranded polymerof deoxyribonucleotide or ribonucleotide bases read from the 5' to the3' end. It includes both self-replcating plasmids, infectious polymersof DNA or RNA and non-functional DNA or RNA. The term "modifiedoligonucleotides" refers to those nucleotides having one or morenucleotides replaced by zwitterionic nucleotides.

The term "complementary" means that one nucleic acid hybridizesselectively to, another nucleic acid. Selectivity of hybridizationexists when hybridization (or base pairing) occurs that is moreselective than total lack of specificity. Typically, selectivehybridization will occur when there is at least about 55% paired basesover a stretch of at least 14-25 nucleotides, preferably at least about65%, more preferably at least about 75%, and most preferably at leastabout 90%. See, M. Kanehisa Nucleic Acids Res. 12:203 (1984),incorporated herein by reference.

As used herein, the term "alkyl" refers to a saturated hydrocarbonradical which may be straight-chain or branched-chain (for example,ethyl, isopropyl, t-amyl, or 2,5-dimethylhexyl). When "alkyl" is used torefer to a linking group, it is taken to be a group having two availablevalences for covalent attachment, for example, --CH₂ CH₂ --, --CH₂ CH₂CH₂ --, --CH₂ CH₂ CH(CH₃)CH₂ -- and --CH₂ (CH₂ CH₂)₂ CH₂ --. Preferredalkyl groups as substituents are those containing 1 to 10 carbon atoms,with those containing 1 to 6 carbon atoms being particularly preferred.Preferred alkyl groups as linking groups are those containing 1 to 10carbon atoms, with those containing 3 to 6 carbon atoms beingparticularly preferred. The term "unsaturated alkyl" refers to alkylgroups having one or more double bonds or triple bonds. When"unsaturated alkyl" is used to refer to a linking group, it is taken tobe a group having two available valences for covalent attachment, forexample, --CH═CHCH₂ --, --C.tbd.CCH₂ --, --CH₂ CH═C(CH₃)CH₂ -- and--C.tbd.C--(CH₂ CH₂)₂ CH₂ --.

The terms "dialkyl ether" and "dialkylthioether" when used to refer tolinking groups refers to such radicals as --CH₂ OCH₂ --, --CH₂ OCH₂ CH₂--, --CH₂ CH₂ CH₂ OCH₂ --, --CH₂ CH₂ OCH₂ CH₂ --, --CH₂ SCH₂ --, --CH₂SCH₂ CH₂ --, --CH₂ CH₂ CH₂ SCH₂ -- and --CH₂ CH₂ SCH₂ CH₂ --, and theirbranched-chain counterparts.

The term "cationic moiety" refers to a group which carries a positivecharge, for example, ammonium, mono-, di- or trialkylammonium,dialkylsulfonium and trialkylphosphonium.

The term "effective binding amount," refers to an amount of modifiedoligonuclectides sufficient to form a duplex with a targetoligonucleotide and either elicit a desired therapeutic response or,when the modified oligonucleotide is coupled with an imaging agent,provide sufficient signal for diagnostic purposes.

The term "chimeric duplex" refers to duplex DNA which is formed from onestrand of natural DNA and a modified oligonucleotide.

As used herein, all numerical ranges are meant to be inclusive of theirupper and lower limits.

Description of the Invention

In one aspect, the present invention provides modified oligonucleotidesof formula I. ##STR1##

In this formula, n is an integer from 4 to 30, inclusive of the upperand lower limits. Preferably, n is an integer of from 4 to 15

The symbol B represents a variety of structures which may be the same ordifferent within each oligonucleotide. Some of these structures arerepresented by formulas II-V: ##STR2## in which X is a linking groupwhich is C₁ -C₁₀ alkyl, C₁ -C₁₀ unsaturated alkyl, dialkyl ether ordialkylthioether; and each Y may be the same or different and is acationic moiety which is --(NH₃)⁺, --(NH₂ R¹)⁺, --(NHR¹ R²)⁺, --(NR¹ R²R³)⁺, dialkylsulfonium or trialkylphosphonium; and R¹, R², and R³ areeach independently lower alkyl having from one to ten carbon atoms.Preferred linking groups for X are C₁ -C₁₀ alkyl and C₁ -C₁₀ unsaturatedalkyl. Particularly preferred linking groups for X are C₃ -C₆ alkyl andC₃ -C₆ unsaturated alkyl. Preferred groups for Y are --(NH₃)⁺, --(NH₂R¹)⁺, --(NHR¹ R²)⁺, --(NR¹ R² R³)⁺, with --(NH₃)⁺ being particularlypreferred.

Other groups for B include adenine (A), guanine (G), thymine (T) andcytosine (C), with the provision that when n is from 4 to 8, no morethan 30% of Bs are A, G, C or T, and when n is from 9 to 30, no morethan 50% of Bs are A, G, C or T. Preferred groups for B include formulasII and III, and A, G, C and T.

Another aspect of the present invention resides in compositions forbinding to an RNA, a DNA, a protein or a peptide. These compositionscontain a modified oligonucleotide of the present invention which ispresent in an effective amount for binding to RNA, DNA, a protein or apeptide, along with an acceptable sterile pharmaceutical carrier.

Still another aspect of the present invention resides in a method forforming chimeric duplexes and triplexes between zwitterionicoligonucleotides and natural DNA. In this method, natural DNA is treatedwith a complementary modified oligonucleotide of formula I. ##STR3##

In this formula, n is an integer from 4 to 30 and preferably, n is aninteger of from 4to 15.

The symbol B represents a variety of structures which may be the same ordifferent within each oligonucleotide. Some of these structures arerepresented by formulas II-V: ##STR4## in which X is a linking groupwhich is C₁ -C₁₀ alkyl, C₁ -C₁₀ unsaturated alkyl, dialkyl ether ordialkylthioether; and each Y may be the same or different and is acationic moiety which is --(NH₃)⁺, --(NH₂ R¹)⁺, --(NHR¹ R²)⁺, --(NR¹ R²R³)⁺, dialkylsulfonium or trialkylphosphonium; and R¹, R², and R³ areeach independently lower alkyl having from one to ten carbon atoms.Preferred linking groups for X are C₁ -C₁₀ alkyl and C₁ -C₁₀ unsaturatedalkyl. Particularly preferred linking groups for X are C₃ -C₆ alkyl andC₃ -C₆ unsaturated alkyl. Preferred groups for Y are --(NH₃)⁺, --(NH₂R¹)⁺, --(NHR¹ R²)⁺, --(NR¹ R² R³)⁺, with --(NH₃)⁺ being particularlypreferred.

Other groups for B include adenine (A), guide (G), thymine (T) andcytosine (C), with the provision that when n is from 4 to 8, no morethen 30% of Bs are A, G, C or T, and when n is from 9 to 30 no more than50% of Bs are A, G, C or T. Preferred groups for B include formulas IIand III, and A, G, C and T.

In all aspects of the present invention, preferred positions for thezwitterionic nucleotide monomers are near or at the 5'- and 3'-terminalof the modified oligonucleotide.

The modified oligonucleotides used in the present invention may beprepared from naturally occurring nucleotides and zwitterionicnucleotides, using conventional techniques. Some preferred zwitterionicnucleotides can be prepared using synthetic methods known to one ofskill in the art and described in Hashimoto, et al., J. Org. Chem.58:4194-4195 (1993) and Hashimoto, et al., J. Am. Chem. Soc.115:7128-7134 (1993), incorporated herein by reference. Briefly,zwitterionic uridine and cytidine analogs are synthesized as theirphosphoramidite esters beginning with the corresponding5-iodo-2'-deoxyuridine and 5-iodo-2'-deoxycytidine. Reaction of theiodo-nucleosides with an appropriately protected amino alkyne in thepresence of a palladium catalyst provides the desired carbon frameworkfor further elaboration. Hydrogenation of the newly introduced alkynecan be accomplished over a palladium on carbon catalyst to provideanalogs having a protected amine which is linked to the nucleotide via asaturated carbon tether. In other embodiments the alkyne may kept aspart of the linking group or may be reduced to an alkene usingcontrolled hydrogenation over palladium on carbon catalysts. Theremaining steps to the phosphoramidite esters involve the addition ofvarious protecting groups which are selected for their compatibilitywith subsequent oligonucleotide synthesis. Such protecting groups arewell known to one of skill in the art and conditions and references forparticular procedures are found in Greene and Wuts, Protecting Groups inOrganic Synthesis, Wiley-Interscience, Second Edition, (1991),incorporated herein by reference.

The oligonucleotides of the present invention may be synthesized insolid phase or in solution. Generally, solid phase synthesis ispreferred. Detailed descriptions of the procedures for solid phasesynthesis of oligonucleotides by phosphite-triester, phosphotriester,and H-phosphonate chemistries are widely available. See, for example,Itakura, U.S. Pat. No. 4,401,796; Caruthers, et al., U.S. Pat. Nos.4,458,066 and 4,500,707; Beaucage, et al., Tetrahedron Lett.,22:1859-1862 (1981); Matteucci, et al., J. Am. Chem. Soc., 103:3185-3191(1981); Caruthers, et al., Genetic Engineering, 4:1-17 (1982); Jones,chapter 2, Atkinson, et al., chapter 3, and Sproat, et al., chapter 4,in Oligonucleotide Sygmhsis: A Practical Approach, Gait (ed.), IRLPress, Washington D.C. (1984); Froehler, et al., Tetrahedron Lett.,27:469-472 (1986); Froehler, et al., Nucleic Acid Res., 14:5399-5407(1986); Sinha, et al., Tetrahedron Lett., 24:5843-5846 (1983); andSinha, et al., Nucl. Acids Res., 12:4539-4557 (1984) which areincorporated herein by reference.

Generally, the timing of delivery and concentration of zwitterionicmonomeric nucleotides utilized in a coupling cycle will not differ fromthe protocols typical for unmodified commercial phosphoramidites used incommercial DNA synthesizers. In these cases, one may merely add thesolution containing the protected zwitterionic monomers to a receptacleon a port provided for an extra phosphoramidite on a commercialsynthesizer (e.g., model 380D, Applied Biosystems, Foster City, Calif.,U.S.A.). However, where the coupling efficiency of a particularprotected zwitterionic monomer is substantially lower than the otherphorphoramidites, it may be necessary to alter the timing of delivery orthe concentration of the reagent in order to optimize the synthesis.Means of optimizing oligonucleotide synthesis protocols to correct forlow coupling efficiencies are well known to those of skill in the art.Generally one merely increases the concentration of the reagent or theamount of the reagent delivered to achieve a higher coupling efficiency.Methods of determining coupling efficiency are also well known. Forexample, where the 5'-hydroxyl protecting group is dimethoxytrityl DMT),coupling efficiency may be determined by measuring the DMT cationconcentration during the acidic removal of the DMT group. DMT cationconcentration is usually determined by spectrophotometrically monitoringthe acid to wash. The acid/DMT solution is a bright orange color.Alternatively, since capping prevents further extension of anoligonucleotide where coupling has failed, coupling efficiency may beestimated by comparing the ratio of truncated to full lengtholigonucleotides utilizing, for example, capillary electrophoresis orHPLC.

Solid phase oligonucleotide synthesis may be performed using a number ofsolid supports. A suitable support is one which provides a functionalgroup for the attachment of a protected monomer which will become the 3'terminal base in the synthesized oligonucleotide. The support must beinert to the reagents utilized in the particular synthesis chemistry.Suitable supports are well known to those of skill in the art. Solidsupport materials include, but are not limited to polacryloylmorpholide,silica, controlled pore glass (CPG), polystyrene, polystyrene/latex, andcarboxyl modified teflon. Preferred supports are amino-functionalizedcontrolled pore glass and carboxylfunctionalized teflon.

Solid phase oligonucleotide synthesis requires, as a starting point, afully protected monomer (e.g., a protected nucleoside) coupled to thesolid support. This coupling is typically through the 3'-hydroxyl.Typically, a linker group is covalently bound to the 3'-hydroxyl on oneend and covalently bound to the solid support on the other end. Thefirst synthesis cycle then couples a nucleotide monomer, via its3'-phosphate, to the 5'-hydroxyl of the bound nucleoside through acondensation reaction that forms a 3'-5' phosphodiester linkage.Subsequent synthesis cycles add nucleotide monomers to the 5'-hydroxylof the last bound nucleotide. In this manner an oligonucleotide issynthesized in a 3' to 5' direction producing a "growing"oligonucleotide with its 3' terminus attached to the solid support.

Numerous means of linking nucleoside monomers to a solid support areknown to those of skill in the art, although monomers covalently linkedthrough a succinate or hemisuccinate to controlled pore glass aregenerally preferred. Conventional protected nucleosides coupled througha hemisuccinate to controlled pore glass are commercially available froma number of sources (e.g. Glen Research, Sterling, Vt., U.S.A.; AppliedBiosystems, Foster City, Calif., U.S.A.; and Pharmacia LKB, Piscataway,N.J. U.S.A.).

Placement of a modified (protected zwitterionic) nucleotide at the 3'end of an oligonucleotide requires initiating oligonucleotide synthesiswith a fully blocked furanosyl modified nucleotide linked to the solidsupport. In a preferred embodiment, linkage of the modified nucleosideis accomplished by first derivatizing the modified nucleotide as ahemisuccinate. The hemisuccinate may then be attached to aminofunctionalized controlled pore glass in a condensation reaction usingmesitylene-2-sulfonyl chloride/1methyl-1H-imidazole as a condensingagent. Controlled pore glass functionalized with a number of differentreactive groups is commercially available (e.g., Sigma Chemical, St.Louis, Mo., U.S.A.). A similar coupling scheme is described by Atkinson,et al., chapter 3 in Oligonucleotide Synthesis: A Practical Approach,Gait (ed.), IRL Press, Washington, D.C., (1984).Triisopropylbenzenesulfonyl chloride, imidazolides, triazolides or eventhe tetrazolides may also be used as condensing agent.Dicyclohexylcarbodiimide (DCC) and structural analogs are also suitablelinkers. Other linkers and appropriate condensing groups are well knownto those of skill in the art.

Once the full length oligonucleotide is synthesized, the oligonucleotideis deprotected and cleaved from the solid support prior to use. Cleavageand deprotection may occur simultaneously or sequentially in any order.The two procedures may be interspersed so that some protecting groupsare removed from the oligonucleotide before it is cleaved off the solidsupport and other groups are deprotected from the cleavedoligonucleotide in solution. The sequence of events depends on theparticular blocking groups present, the particular linkage to a solidsupport, and the preferences of the individuals performing thesynthesis. Where deprotection precedes cleavage, the protecting groupsmay be washed away from the oligonucleotide which remains bound on thesolid support. Conversely, where deprotection follows cleavage, theremoved protecting groups will remain in solution with theoligonucleotide. Often the oligonucleotide will require isolation fromthese protecting groups prior to use.

In a preferred embodiment, and most commercial DNA syntheses, theprotecting group on the 5'-hydroxyl is removed at the last stage ofsynthesis. The oligonucleotide is then cleaved off the solid support,and the remaining deprotection occurs in solution. Removal of the5'-hydroxyl protecting group typically requires treatment with the samereagent utilized throughout the synthesis to remove the terminal5'-hydroxyl groups prior to coupling the next nucleotide monomer. Wherethe 5'-hydroxyl protecting group is a dimethoxytrityl group,deprotection can be accomplished by treatment with acetic acid,dichloroacetic acid or trichloroacetic acid.

Typically, both cleavage and deprotection of the exocyclic amines areeffected by first exposing the oligonucleotide attached to a solid phasesupport (via a base-labile bond) to the cleavage reagent for about 1-2hours, so that the oligonucleotide is released from the solid support,and then heating the cleavage reagent containing the releasedoligonucleotide for at least 20-60 minutes at about 80-90° C. so thatthe protecting groups attached to the exocyclic amines are removed. Thedeprotection step may alternatively take place at a lower temperature,but must be carried out for a longer period of time (e.g., the heatingcan be at 55° C. for 5 hours). In general, the preferred cleavage anddeprotection reagent is concentrated ammonia.

Where the oligonucleotide is a ribonucleotide and the 2'-hydroxy groupis blocked with a tert-butyldimethylsilyl (TBDMS) moiety, the lattergroup may be removed using tetrabutylammonium fluoride intetrahydrofuran at the end of synthesis. See Wu, et al., J. Org. Chem.55:4717-4724 (1990). Phenoxyacetyl protecting groups can be removed withanhydrous ammonia in alcohol (under these conditions the TBDMS groupsare stable and the oligonucleotide is not cleaved). The benzoylprotecting group of cytidine is also removed with anhydrous ammonia inalcohol.

Cleaved and fully deprotected oligonucleotides may be used directly(after lyophilization or evaporation to remove the deprotection reagent)in a number of applications, or they may be purified prior to use.Purification of synthetic oligonucleotides is generally desired toisolate the full length oligonucleotide from the protecting groups thatwere removed in the deprotection step and, more importantly, from thetruncated oligonucleotides that were formed when oligonucleotides thatfailed to couple with the next nucleotide monomer were capped duringsynthesis.

Oligonucleotide purification techniques are well known to those of skillin the art. Methods include, but are not limited to, thin layerchromatography (TLC) on silica plates, gel electrophoresis, sizefractionation (e.g., using a Sephadex column), reverse phase highperformance liquid chromatography (HPLC) and anion exchangechromatography (e.g., using the mono-Q column, Pharmacia-LKB,Piscataway, N.J. U.S.A.). For a discussion of oligonucleotidepurification see McLaughlin, et al., chapter 5, and Wu, et al., chapter6 in Oligonucleotide Synthesis: A Practical Approach, Gait (ed.), IRLPress, Washington, D.C., (1984).

When the modified oligonucleotides of the present invention are to beused for imaging purposes, the desired label (e.g., radiolabel,fluorescent tag, biotin) can be attached by means well known to those ofskill in the art and described in Telser, et al., J. Am. Chem. Soc.111:7221-7226 and 7226-7232 (2989); Allen, et al., Biochemistry28:4601-4607 (2989); Smith, et al., Nucleic Acids Res 13:2399-2412(1985); Haralambidis, et al., ibid., 15:4857-4876 (1987); and Gebeyehu,et al., ibid., 15:4513-4535 (1987, the disclosures of which areincorporated herein by reference.

In preferred embodiments, the modified oligonucleotides of the presentinvention can be used to bind to DNA or RNA. The DNA or RNA sequencesmay be present in a variety of cells including normal cells, neoplasticcells, prokaryotic or eukaryotic cells, and a virus. The sequences maybe bacterial sequences, plasmid sequences, viral sequences, chromosomalsequences, mitochondrial sequences, or plastid sequences. The targetsequences may have both open reading frames for coding proteins anduntranslated portions. The target sequences may therefore be involved ininhibiting the expression of a particular protein or enhancing theexpression of a particular protein by inhibiting the expression of arepressor. Additionally, the target sequences may be involved inreducing the proliferation of viruses or neoplastic cells.

The modified oligonucleotides may be used in vitro or in vivo formodifying the phenotype of cells, or for limiting the proliferation ofpathogens such as viruses, bacteria, protists, Mycoplasma species,Chlamydia or the like, or for inducing morbidity in neoplastic cells orspecific classes of normal or diseased cells. Thus, the modifiedoligonucleotides may be administered to a host which is subject to or ina diseased state. When administered to a host, the oligonucleotides maybe used to treat infection by a variety of pathogens, for example,enterotoxigenic bacteria, Pneumococci, Neisseria organisms, Giardiaorganisms, and Entamoebas. The modified oligonuclectides may also beused as cytotoxic or cytostatic agents for neoplastic cells, such ascarcinoma cells, sarcoma cells, and lymphoma cells. The oligonucleotidesmay be used to modulate the function of immune system cells such asspecific B-cells; specific T-cells, such as helper cells, suppressorcells, cytotoxic T-lymphocytes (C), and natural killer (NX) cells.Modulation of immune function can be useful in treatment of a variety ofdiseases such as cancer and immune system disease.

The modified oligonuclectides may be selected so as to be capable ofinterfering with transcription product maturation or expression ofproteins by any of the mechanisms involved with the binding of themodified oligonucleotide to its target IC sequence. These mechanisms mayinclude interference with processing, inhibition of transport across thenuclear membrane, cleavage by endonucleases, or the like.

The modified oligonucleotides may be complementary to nucleic acidsequences such as those encoding growth factors, lymphokines,immunoglobulins, T-cell receptor sites, MHC antigens, DNA or RNApolymerases, antibiotic resistance, multiple drug resistance (mdr),genes involved with metabolic processes, such as the formation of aminoacids, nucleic acids, or the like. The modified oligonucleotides may becomplementary to nucleic acid sequences including introns or flankingsequences associated with the open reading fines.

The modified.oligonucleotides of the present invention may be used inthe treatment of infectious diseases, cancers, autoimmune diseases andconditions associated with organ transplants. In the treatment ofinfectious diseases, the target sequences include those genes associatedwith AIDS, CMV, herpes, drug resistance plasmids, and trypanosomes. Inthe treatment of cancer, the target sequences can be DNA or RNAassociated with oncogenes, tumor suppressor genes, and related genes.Additionally, the modified oligonucleotides may also target genesassociated with drug resistance and their gene products. For thetreatment of autoimmune diseases, the modified oligonucleotides can be,for example, target sequences associated with rheumatoid arthritis, TypeI diabetes, systemic lupus and multiple sclerosis.

In addition to binding nucleic acids, the modified oligonucleotides ofthe present invention may also be employed for binding to proteinsincluding, but not limited to, ligands, receptors, and/or enzymes,whereby the modified oligonucleotides inhibit the activity of theproteins.

The modified oligonucleotides used in the present inventive method maybe administered in any suitable manner, preferably with pharmaceuticallyacceptable carriers. One skilled in the art will appreciate thatsuitable methods of administering such compounds in the context of thepresent invention to an animal are available, and, although more thanone route can be used to administer a particular compound, a particularroute can provide a more immediate and more effective reaction thananother route. Pharmaceutically acceptable carriers are also well-knownto those who are skilled in the art. The choice of carrier will bedetermined in part by the particular modified oligonucleotide, as wellas by the particular method used to administer the composition.Accordingly, there is a wide variety of suitable formulations of thepharmaceutical composition of the present invention.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the modified oligonucleotidedissolved in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as solids, granules or gelatin; (c) suspensions in anappropriate liquid; and (d) suitable emulsions. Tablet forms can includeone or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates,corn starch, potato starch, tragacanth, microcrystalline cellulose,acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc,magnesium sterate, stearic acid, and other excipients, colorants,fillers, binders, diluents, buffering agents, mositening agents,preservatives, flavoring agents, dyes, disintegrating agents, andpharmaceutically compatible carriers Lozenge forms can comprise theactive ingredient in a flavor, usually sucrose and acacia or tragacanth,as well as pastilles comprising the active ingredient in an inert base,such as gelatin and glycerin or sucrose and acacia emulsions, gels, andthe like containing, in addition to the active ingredient, such ascarriers as are known in the art.

The active ingredient, alone or in combination with other suitablecomponents, can be made into aerosol formulations to be administered viainhalation. These aerosol formulations can be placed into pressurizedacceptable propellants, such as dichloro difluoromethane, propane,nitrogen, and the like.

Suitable formulations for rectal administration include, for example,suppositories, which consist of the active ingredient with a suppositorybase. Suitable suppository bases include natural or synthetictriglycerides or paraffin hydrocarbons. In addition, it is also possibleto use gelatin rectal capsules which consist of a combination of theactive ingredient with a base, such as, for example, liquidtriglycerides, polyethylene glycols, and paraffin hydrocarbons.

Formulations suitable for parenteral administration, such as, forexample, by intravenous, intramuscular, intradermal, intraperitoneal,and subcutaneous routes, include aqueous and non-aqueous, isotonicsterile injection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. The formulations can bepresented in unit-dose or multi-dose sealed containers, such as ampulesand vials, and can be stored in a freeze-dried lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for example,water, for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described.

The dose administered to an animal, particularly a human, in the contextof the present invention should be sufficient to effect a therapeuticresponse in the animal over a reasonable time frame. The dose will bedetermined by the strength of the particular compound employed and thecondition of the animal, as well as the body weight or surface area ofthe animal to be treated. The size of the dose also will be determinedby the existence, nature, and extent of any adverse side-effects thatmight accompany the administration of a particular compound in aparticular animal.

In the practice of this invention, the modified oligonucleotides can beadministered, for example, by intravenous infusion, orally, topically,intraperitoneally, intravesically or intrathecally.

Additionally, the modified oligonucleotides of the present invention maybe administered encapsulated in liposomes, pharmaceutical compositionswherein the active ingredient is contained either dispersed or variouslypresent in corpuscles consisting of aqueous concentric layers adherentto lipidic layers. The active ingredient, depending upon its solubility,may be present both in the aqueous layer and in the lipidic layer, or inwhat is generally termed a liposomic suspension. The hydrophobic layer,generally but not exclusively, comprises phospholipids such as lecithinand sphingomycelin, steroids such as cholesterol, more or less ionicsurfactants such as dicetylphosphate, stearylamine, or phosphatidicacid, and/or other materials of a hydrophobic nature. The diameters ofthe liposomes generally range from about 15 nm to about 5 microns

For oral administration, modified oligonucleotides of the presentinventive method can be administered at the rate up to 300 mg/m² bodysurface area, which approximates 6 grams/day in the average patient. Apreferred rate is from 1 to 300 mg/m² body surface area. This can beaccomplished via single or divided doses. For intravenousadministration, such compounds can be administered at the rate of up toabout 2500 mg/m² /d, preferably from about 0.1 to about 200 mg/m/d. Forintravesicle administration, such compounds can be administered at therate of up to about 2500 mg/m² /d, preferably from about 0.1 to about200 mg/m² /d. For topical administration, the rate can be up to about2500 mg/m² /d, preferably from about 0.1 to about 200 mg/m² /d. The dosefor inhalation/aerosol administration can be up to about 2500 mg/m² /d,preferably from about 0.1 to about 200 mg/m² /d. Direct intraperitonealadministration can be performed using up to about 3000 mg/m² /d,preferably from about 0.1 to about 100 mg/m² /d. The dose for reservoiradministration to the brain or spinal fluid can be up to about 2000mg/m² /d, preferably from about 0.1 to about 100 mg/m² /d. For slowrelease intraperitoneal or subcutaneous administration, the dose can befrom about 0.1 to about 5000 mg/day in a bolus, preferably from about1.0 to about 200 mg/day. For intrathecal administration, the dose can beup to about 2000 mg/m² /d, preferably from about 0.1 to about 100 mg/m²/d.

The following experimental results are offered by way of example and notby way of limitation.

EXAMPLES

General Methods

A air sensitive reactions were carried out under an atmosphere of N₂ orAr. Melting points were taken on a Thomas-Hoover Uni-melt capillarymelting point apparatus and are uncorrected. ¹ NMR spectra were recordedon a GE QE-300 (300 MHz) spectrometer. ¹³ C NMR spectra were recorded oneither a GE QE-300 (75 MHz) or GN-500 (125 MHz) spectrometer. ³¹ P NMRspectra were recorded on a GE GN-500 (202 MHz, H₃ PO₄ external standard)spectrometer. Infrared spectra were taken on a Nicolet 5-DX FT-IRspectrophotometer. Ultraviolet-visible spectra were recorded on aHewlett-Packard 8452A diode-array spectrophotometer. Elemental analyseswere carried out at Desert Analytics Organic Microanalysis Laboratory(Tucson, Ariz.), Mass spectra and exact mass determinations wererecorded on a VG-ZAB2FHF mass spectrometer using FAB ionization at theSouthern California Regional Mass Spectrometry Facility (University ofCalifornia, Riverside). Laser desorption mass spectra were recorded on aFinnegan Lasermat mass spectrometer at the Biotechnology InstrumentationFacility (University of California, Riverside). Chemicals were purchasedfrom either Sigma or Aldrich Chemical Companies (St. Louis, Mo., USA andMilwaukee, Wis., USA, respectively). Solvents were purchased from FisherScientific, AG1X8 (⁻ OH) anion exchange resin was purchased from Bio-Rad(Hercules, Calif., USA). T4 polynucleotide kinase was purchased fromBoehringer Mannheim (Indianapolis, Ind., USA). 5'-(γ-³² P)ATP (˜3000Ci/mmol) was purchased from Amersham (Arlington Heights, Ill., USA).Controlled pore glass support was purchased from CPG, Inc. (Fairfield,N.J., USA). Tetrakis(triphenylphosphine)-palladium (0) was prepared by aliterature method. See, Coulson, D. R. Inorganic Syntheses, 13:121(1971). Pyridine and CH₂ Cl₂ were distilled from CaH₂ and stored overmolecular sieves. Dimethylformamide (DMF) was stored over molecularsieves prior to use.

Two chemicals used in the following syntheses were prepared by methodswell known in the art.

N-trifluoroacetyl-1-amino-5-hexyne

5-Hexyn-1-ol was treated with methanesulfonyl chloride in the presenceof triethylamine and an organic solvent to provide the correspondingmethanesulfonate ester. Displacement of the ester with the sodium saltof trifluoroacetamide (formed from NaH and trifluoroacetamide) providesN-trifluoroacetyl-1-amino-5-hexyne.

N-trifluoroacetyl-1-amino-2-propyne

Treatment of ethyl trifluoroacetate with 1-amino-2-propyne in a suitableorganic solvent, produces N-trifluoroacetyl-1-amino-2-propyne afterremoval of solvent and distillation.

Synthesis

Examples 1-3 provide the syntheses of protected phosphoramiditeprecusors to the zwitterionic uridine analogs 1 and 2, and zwitterioniccytidine analog 3, respectively. Example 4 illustrates the methods usedfor oligonucleotide synthesis. ##STR5##

Example 1

This example illustrates the synthesis of5-(6-N-(trifluoroacetyl)-aminohexyl)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyuridine2-cyanoethyl N,N-diisopropylphosphoramidite, (4) beginning withcommercially available 5-iodo-2'-deoxyuridine.

A. Conversion of 5-iodo-2'-deoxyuridine to5-(6-N-(trifluoroacetyl)-amino-1-hexynyl)-2'-deoxyuridine (5). ##STR6##

To a suspension of 5-iodo-2'-deoxyuridine (650 mg, 1.84 mmol) in CH₂ Cl₂(7.0 mL) was added trifluoroacetic anhydride (2.35 mL, 16.6 mmol) atroom temperature. The mixture was stirred overnight. After concentrationof the mixture, the residue was dried in vacuo at room temperature togive as a solid foam 1.30 g of3',5'-di-O-trifluoroacetyl-5-iodo-2'-deoxyuridine.

To a mixture of di-O-trifluoroacetyl-5-iodo-2'-deoxyuridine,H-trifluoroacetyl-1-amino-5-hexyne (1.066 g, 5.52 mmol),tetrakis(triphenylphosphine)palladium (0) (150 mg, 0.13 mmol), andcopper (I) iodide (50 mg, 0.26 mmol) were added dry DMF (10 mL) and Et₃N (0.768 mL, 5.52 mmol). The mixture was stirred at room temperature for24 h, and then concentrated under a vacuum. The residue was purified byflash chromatography (SiO₂, MeOH (5%-10%)/CH₂ Cl₂). The fractioncontaining 5 was concentrated and treated with anion exchange resinAG1X8 (HCO₃ ⁻, 1.58 g, 3.0 eq.) in 5 mL of 1/1, CH₂ Cl₂ /MeOH at roomtemperature for 30 min. Evaporation of the solvent gave 5: white solid,605 mg, 78% yield; mp 154-157° C. (MeOH/Et₂ O); ¹ H NMR (DMSO-d6) δ 1.48(m, 2 H), 1.58 (m, 2 H), 2. 10 (m, 2 H), 2.38 (t, 2 H, J=6.9 Hz), 3.20(m, 2 H), 3.57 (m, 2 H 3.77 (m, 1 H), 4.21 (m, 1 H), 5.08 (t, 1 H, J=4.6Hz), 5.24 (d, 1 H, J=3.8 Hz), 6.10 (t, 1 H, J=6.6 Hz), 8.10 (s, 1 H),9.43 (br s, 1 H), 11.56 (br s, 1 H); ¹³ C NMR (DMSO-d6) δ 18.61, 25.73,27.73, 38.87, 40.20, 61.20, 70.38, 73.30, 84.77, 87.75, 93.06, 99.16,116.19 (CF₃, J_(C-F) =288.9 Hz), 142.96, 149.68, 156.41 (COCF₃, J_(C-F)=36.4 Hz), 162.00; UV (MeOH) nm (e×10³) 230 (11.9), 292 (12.3); IR (KBr)cm⁻¹ 3521, 3407, 3320, 2239, 1697, 1285, 1214, 1195, 1032; MS (FAB⁺)m/z: 420 (MH⁺), 330, 304; HRMS (FAB⁺) calc. for C₁₇ H₂₁ N₃ O₆ F₃420.1382 (MH⁺), found 420.1382; Anal. calc, for C₁₇ H₂₀ N₃ O₆ F₃ : C,48.69; H, 4.81; N, 10.02, found: C, 48.51; H, 4.73; N, 9.92.

B. Conversion of 5 to5-(6-N-(trifluoroacetyl)-aminohexyl)-2'-deoxyuridine (6). ##STR7##

A mixture of 5 (480 mg, 1.14 mmol) and 10% Pd/C (95 mg) in MeOH (6 mL)was stirred under H₂ pressure (50 psi) at room temperature for 3 days.The mixture was filtered through celite and concentrated to give 6: 475mg, 98% yield; mp 136-140° C. (Et₂ O); ¹ H NMR (DMSO-d6) δ 1.2-1.3 (m, 4H), 1.3-1.5 (m, 4 H), 2.07 (m, 2 H), 2.16 (m, 2 H), 3.15 (t, 2 H, J=6.9Hz), 3.56 (m, 2 H), 3.76 (m, 1H), 4.23 (br s, 1 H), 5.02 (br s, 1H),5.23 (br s, 1H), 6.16 (t, 1 H, J=6.8 Hz), 7.68 (s, 1H), 9.39 (br s, 1H),11.22 (br s, 1 H); ¹³ C NMR (DMSO-d6) δ 26.14, 26.44, 28.07, 28.40,28.40, 39.56, 39.98, 61.49, 70.65, 84.10, 87.51, 113.76, 116.20 (CF₃,J_(C-F) =288.5 Hz), 136.33, 150.60, 156.33 (COCF₃, J_(C-F) =35.6 Hz),163.68; UV (MeOH) nm (e×10³) 212 (15.9), 268 (13.6); IR (KBr) cm⁻¹ 3552,3384, 3328, 1701, 1684, 1559, 1203, 1180, 1170; MS (FAB⁺) m/z: 424(MH⁺); HRMS (FAB⁺) calc. for C₁₇ H₂₅ N₃ O₆ F₃ 424.1695 (MH⁺), found424.1706; Anal. calc. for C₁₇ H₂₄ O₆ F₃ : C, 48.23; H, 5.71; N, 9.92,found: C, 48.19; H, 5.68; N, 10.00.

C. Conversion of 6 to5-(6-N-(trifluoroacetyl)-aminohexyl)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyuridine(7). ##STR8##

To a solution of 6 (490 mg, 116 mmol) in dry pyridine (10 mL) was added4,4'-dimethoxytrityl chloride (430 mg, 1.27 mmol) in small portions over30 min. at room temperature. The reaction mixture was stirred at roomtemperature for 2 h. The mixture was then concentrated, and the residuewas purified by flash chromatography (SiO₂, MeOH (3-10%)/pyridine(0.5%)/CH₂ Cl₂) to give 7: white amorphous solid, 753 mg, 89% yield; ¹ HNMR (CDCl₃) δ 1.0-1.1(m, 4 H), 1.15-1.3 (m, 2 H) 1.3-1.4 (m, 2 ), 1.71(m, 1 H), 1.93 (m, 1 H), 2.32 (m, 1 H), 2.41 (m, 1 H), 2.64 (br s, 1 H),3.31 (dd, 1 H, J=2.4 Hz, 10.5 Hz), 3.38 (m, 2 H), 353 (dd, 1 H), J=2.6Hz, 10.5 Hz), 3.79 (s, 6 1), 4.05 (1 H), 4.57 (m, 1 H), 6.44 (dd, 1 H,J=6.2 Hz, 7.5 Hz), 6.71 (br s, 1 H), 6.83 (d, 4 H, J=8.6 Hz), 7.2-7.5(m, 7 H), 7.39 (2 H), 7.57 (s, 1 H), 9.22 (s, 1 H); ¹³ C NMR (CDCl₃) δ25.71, 26.40, 28.22, 28.32, 28.32, 39.90, 40.97, 55.27, 63.50, 72.44,84.70, 86.39, 86.77, 113.27, 115.82, 115.97 (CF₃, J_(C-F) =287.4 Hz),127.14, 127.98, 128.20, 130.11, 135.48, 135.90, 144.30, 150.75, 157.26(COCF₃, J_(C-F) =36.8 Hz), 158.65, 164.10; UV (MeOH) nm (e×10³) 234(22.1), 270 (10.6); IR (KBr) cm⁻¹ 3329, 1701, 1680, 1508, 1252, 1178; MS(FAB⁺) m/z: 726 (ME⁺), 725 (M⁺), 303; HRMS (FAB⁺) calc. for C₃₈ H₄₂₈ N₃O₈ F₃ 725.2924 (M⁺), found 725.2964; Anal. calc. for C₃₈ H₄₂ N₃ O₈ F₃ :C, 62.89; H, 5.83; N, 5.79, found: C, 62.65; H, 5.77; N, 6.05.

D. Conversion of 7 to5-(6-N-(trifluoroacetyl)-aminohexyl)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyuridine2-cyanoethyl N,N-diisopropylphosphoramidite (4). ##STR9##

To a solution of 7 (300 mg, 0.41 mmol) in CH₂ Cl₂ (3.0 mL) was addeddiisopropylethylamine (0.216 mL, 1.24 mmol) and 2-cyanoethylN,N-diisopropylchlorophosphine (0.120 mL, 0.54 mmol) successively atroom temperature. The mixture was stirred for 1 h, followed by dilutionwith 100 mL of Cl₂ Cl₂, extraction with 5% aq. NaHCO₃, drying over Na₂SO₄, and concentration. Purification by flash chromatography (SiO₂,EtOAc(30%)/Et₃ N(1%)/CH₂ Cl₂) gave phosphoramidite 4: 367 mg as amixture of two diastereomers, with a small amount of HPO(OCH₂ CH₂CN)N(iPr)₂ as an inseparable impurity (³¹ P NMR δ 14.28), 88:12 of4:H-phosphonate, yield of 4 93%; ¹ H NMR (CDCl₃) selected signals ofdiastereomers δ: 2.40 (t, 2 H, J=3.2 Hz, --OCH₂ CH₂ CN) 2.62 (t, 2 H,J=3.2 Hz, --OCH₂ CH₂ CN), 3.15 (m, 4 H, CF₃ CONHCH₂ --), 3.79 (s, 3 H,OMe), 3.80 (s, 3 H, OMe), 4.11 (m, 1 H, H₄), 4.16 (m, 1 H, H_(4')),4.6-4.7 (m, 2 H, H_(3')), 6.40 (m, 1 H, H_(1')), 6.43 (m, 1 , H_(1')),6.56 (m, 2 H, CF₃ CONH--); ³¹ P NMR (CDCl₃) δ: 148.57, 148.93; MS (FAB⁺)m/z: 926 (MH⁺), 818, 708, 404, 303; HRMS (FAB⁺) calc. for C₄₇ H₆₀ N₅ O₉F₃ P 926.408 (MH⁺), found 926.4065.

Example 2

This example illustrates synthesis of5-(3-N-(Trifluoroacetyl)-aminopropyl)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyuridine2-cyanoethyl N,N-diispropylphosphoramidite, 8, beginning with5-iodo-2'-deoxyuridine.

A. Conversion of 5-iodo-2'-deoxyuridine to5-(3-N-(Trifluoroacetyl)-amino-1-propynyl)-2'-deoxyuridine (9).##STR10##

To a suspension of 5-iodo-2'-deoxyuridine (1.018 g, 2.82 mmol) in CH₂Cl₂ (14 mL) at 0° C. was added trifluoroacetic anhydride (3.98 mL, 28.24mmol). The mixture was stirred at 0° C. for 2 h and then at roomtemperature for 12 h. After concentration of the mixture, the residuewas dried in vacuo at room temperature to give3',5'-di-O-trifluoroacetyl-5-iodo-2'-deoxyuridine as a solid foam.

To a mixture of 3',5'-di-O-trifluoroacetyl-5-iodo-2'-deoxyuridine,N-trifluoroacetyl-1-amino-2-propyne (1.27 mL, 8.46 mmol)tetrakis(triphenylphosphine)palladium (0) (325 mg, 0.282 mmol), andcopper (I) iodide (107 mg, 0.564 mmol) were added dry DMF (14 mL) andEt₃ N (0.786 mL, 5.64 mmol). The mixture was stirred at room temperaturefor 21 h, and then concentrated under a vacuum. The residue was purifiedby flash chromatography (SiO₂, MeOH (10%)/CH₂ Cl₂). The fractioncontaining the desired product was concentrated and treated with anionexchange resin (AG1X8, HCO₃ ⁻, 2.4 g, 3.0 eq.) in 30 mL of 1/1, CH₂Cl2/MeOH at room temperature for 30 min. Evaporation of the solvent gave9 as a white solid, 747 mg, 70% yield; ¹ H NMR (DMSO-d6) δ 2.18 (m, 2H), 3.1-3.3 (m, 2 H), 3.55 (m, 2 H), 3.78 (m, 1 H), 4.08 (m, 1 H), 5.07(t, 1 H, J=5.0 Hz), 5.22 (d, 1 H, J=4.2 Hz), 6.09 (t, 1 H, J=6.6 Hz),8.18 (s, 1 H), 9.9-10.1 (br s, 1 H, 11.5-11.7 (br s, 1 H); ¹³ C NMR(DMSO-d6) δ 29.46, 40.12, 61.00, 70.21, 75.40, 84.82, 87.51, 87.67,97.68, 115.81 (CF₃, J_(C-F) =288 Hz), 144.18, 149.44, 156.08 (COCF₃,J_(C-F) =37 Hz), 161.62; UV (H₂ O) nm (e×10³) 232 (4.15), 288 (4.53); IR(KBr) cm⁻¹ 3482, 2950, 1700, 1476, 1304, 1214, 1156, 1100; MS (FAB⁺)m/z: 378 (MH⁺), 262; HRMS (FAB⁺) calc. for C₁₄ H₁₅ N₃ O₆ F₃ 378.0913(MH⁺), found 378.0898.

B. Conversion of 9 to5-(3-N-Trifluoroacetyl)-aminopropyl-2'-deoxyuridine (10). ##STR11##

A mixture of 5-(3-N-(trifluoroacetyl)-amino-1-propynyl)-2'-deoxyuridine(9, 733 mg, 1.94 mmol) and 10% Pd/C (206 mg) in MeOH (25 mL) was stirredunder H₂ pressure (50 psi) at room temperature for 3 days. The mixturewas filtered through celite and concentrated to give the desiredproduct, 10: 703 mg, 95% yield; mp 155-158° C. (CH₃ CN-hexane); ¹ H NMR(DMSO-d6) δ 1.62 (m, 2 H), 2.08 (m, 2 H), 2.19 (m, 2 H), 3.16 (m, 2 H),3.55 (m, 2 H), 3.75 (m, 1 H), 4.23 (br s, 1 H), 5.00 (br s, 1 H), 5.22(br d, 1 H, J=2.8 Hz), 6.15 (t, 1 H, J=6.7 Hz), 7.67 (s, 1 H), 9.39 (brs, 1 H), 11.28 (br s, 1 H); ¹³ C NMR (DMSO-d6) δ 24.01, 27.31, 38.88,39.63, 61.49, 70.61, 84.14, 87.50, 112.81, 116.16 (CF₃, J_(C-F) =288Hz), 136.65, 150.50, 156.35 (COCF₃, J_(C-F) =36 Hz), 163.54; UV (MeOH)nm (e×10³) 268 (7.4); IR (KBr) cm⁻¹ 3432, 3320, 1734, 1696, 1654, 1274,1204, 1185, 1153; MS (FAB⁺) m/z: 382 (MH⁺), 266; HRMS (FAB⁺) calc. forC₁₄ H₁₉ N₃ O₆ F₃ 382.1226 (MH⁺), found 382.1213; Anal. calc. for C₁₄ H₁₈N₃ O₆ F_(3:) C, 44.10; H, 4.76; N, 11.02, found: C, 43.86; H, 4.77; N,10.75.

C. Conversion of 10 to5-(3-(Trifluoroacetyl)-aminopropyl)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyuridine(11). ##STR12##

To a solution of 5-(3-N-(trifluoroacetyl)-aminopropyl)-2'-deoxyuridine(10, 100 mg, 0.262 mmol) in dry pyridine (2 mL) was added4,4'-dimethoxytrityl chloride (90 mg, 0.266 mmol) at room temperature.The reaction mixture was stirred at room temperature for 3 h. Themixture was then concentrated, and the residue was purified by flashchromatography (SiO₂, MeOH (3-5%)/pyridine (0.5%)/CH₂ Cl₂) to give thedesired product, 11, as a white amorphous solid: 154 mg, 86% yield; ¹ HNMR (CDCl₃) δ 1.35 (m, 2 H), 1.69 (m, 1 H), 1.87 (m, 1 H), 2.11 (br s, 1H, 2.3-2.5 (m, 2 H) 3.06 (m, 2 H), 3.37 (m, 1 H), 3.55 (m, 1 H), 3.80(s, 6 H), 4.06 (m, 1 H), 4.62 (m, 1 H), 6.44 (t, 1 H, J=6.7 Hz), 6.84(d, 4 H, J=8.5 Hz), 7.2-7.4 (m, 10 H), 7.65 (s, 1 H), 8.5 (br, s 1 H);¹³ C NMR (CDCl₃)δ23.34, 28.10, 38.59, 40.99, 55.32, 63.59, 72.65, 84.92,86.92, 86.98, 113.35, 114.09, 115.99 (CF₃, J_(C-F) =288 H), 127.36,128.06, 128.29, 130.17, 130.23, 135.29, 137.41, 144.15, 150.56, 157.27(COCF₃, J_(C-F) =37 Hz), 158.82, 164.62; UV (MeOH) nm (e×10³) 232(19.2), 270 (9.2); IR (KBr) cm⁻¹ 3305, 1711, 1676, 1509, 1252, 1177; MS(FAB⁺) m/z: 683 (M⁺), 303; HRMS (FAB⁺) calc. for C₃₅ H₃₆ N₃ O₈ F₃683.2455 (M⁺), found 683.2437; Anal. calc. for C₃₅ H₃₆ N₃ O₈ F₃ : C,61.49; H, 5.31; N, 6.15, found: C, 61.17; H, 5.34; N, 5.85.

D. Converison of 11 to5-(3-N-(Trifluoroacetyl)-aminopropyl)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyuridine2-cyanoethyl N,N-diisopropylphosphoramidite (8). ##STR13##

To a solution of5-(3-N-(trifluoroacetyl)-aminopropyl)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyuridine(11, 190 mg, 0.278 mmol) in CH₂ Cl₂ (3.0 mL) was addeddiisopropylethylamine (0.145 mL, 0.832 mmol) and 2-cyanoethylN,N-diisopropylchlorophosphine (0.080 mL, 0.36 mmol) successively atroom temperature. The mixture was stirred for 1 h, diluted with 100 mLof CH₂ Cl₂, extracted with 5% aq. NaHCO₃, dried over Na₂ SO₄, andconcentrated. The residue was purified by flash chromatography (SiO₂,MeOH(3%)/Et₃ N(1%)/CH₂ Cl₂) to provide the desired phosphoramidite, 8 asa mixture of two diastereomers (264 mg), with a small amount of HPO(OCH₂CH₂ CN)N(iPr)₂ as an inseparable impurity (³¹ P NMR δ 14.28), 75:25 ofphosphoramidite:H-phosphonate, yield of phosphoramidite: 99%; ¹ H NMR(CDCl₃) selected signals of diastereomers δ: 1.6-1.7 (m, 2 H, CF₃CONHCH₂ CH₂ CH₂ --), 1.8-1.9 (m, 2 H, CF₃ CONHCH₂ CH₂ CH₂ --), 2.41 (t,2 H, J=3.2 Hz, --OCH₂ CH₂ CN), 2.62 (t, 2 H, J=3.2 Hz), --OCH₂ CH₂ CN),3.0-3.1 (m, 4 H, CF₃ CONHCH₂ --), 3.79 (s, 6 H, OMe), 3.80 (s, 6 H,OMe), 4.14 (m, 1 H, H₄ '), 4.29 (m, 1 H, H₄ '), 4.7 (m, 2 H, H_(3')),6.40-6.48 (m, 2 H, H₁ '), 7.64 (s, 1 H, H₆), 7.68 (s, 1 H, H₆); ³¹ P NMR(CDCl₃)δ: 148.66, 148.90; MS (FAB⁺) m/z: 884 (MH⁺), 666, 362, 303; HRMS(FAB⁺) calc. for C₄₄ H₅₄ N₅ O₉ F₃ P 884.3611 (MH⁺), found 884.3640.

Example 3

This example illustrates the synthesis of5-(6-N-(trifluoroacetyl)-aminohexyl)-4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine2-cyanoethyl N,N-diisopropyl phosphoramidite, 12, beginning withcommercially available 5-iodo-2'-deoxycytidine.

A. Conversion of 5-iodo-2'-deoxycytidine to5-(6-N-(trifluoroacetyl)-amino-1-hexynyl)-2'-deoxycytidine (13).##STR14##

To 5-iodo-2'-deoxycytidine (1.496 g, 4.24 mmol),tetrakis(triphenyl-phosphine)palladium (0) (490 mg, 0.42 mmol), copper(I) iodide (163 mg, 0.86 mmol) and N-trifluoroacetyl-1-amino-5-hexyne(2.455 g, 12.7 mmol) were added DMF (21 mL) and Et₃ N (1.2 mL, 8.63mmol). After stirring for 12 h, AG-1X8 anion exchange resin (HCO₃ ⁻form, 3.0 eq) 20 mL MeOH and 20 mL CH₂ Cl₂ were added, and thesuspension was stirred for 1 h. The reaction was filtered through asintered glass funnel, and the DMF was removed in vacuo. Flashchromatography (SiO₂, MeOH (10%-20%)/CH₂ Cl₂) provided 13: 1.389 g, 78%yield; mp 146-148° C. dec. EtOAc); ¹ H NMR (DMSO-d6) δ 1.55 (m, 4 H),1.96 (m, 1 H), 2.11 (ddd, 1 H, J=3.5, 5.7, 13.0 Hz), 2.41 (t, 2 H, J=6.5Hz), 3.20 (m, 2 H), 3.56 (m, 2 H), 3.77 (m, 1 H), 4.18 (m, 1 H), 5.03(t, 1 H, J=5.0 Hz), 5.18 (d, 1 H, J=4.1 Hz), 6.10 (t, 1 H, J=6.5 Hz),6.71 (br s, 1 H), 7.67 (br s, 1 H), 8.05 (s, 1 H), 9.42 (m, 1 H); ¹³ CNMR (DMSO-d6) δ 18.67, 25.26, 27.61, 38.73, 40.73, 61.06, 70.18, 72.27,85.26, 87.41, 90.40, 95.31, 115.99 (CF ₃, J_(C-F) =288.6 Hz), 143.55,153.52, 156.21 (COCF₃, J_(C-F) =35.6 Hz), 164.41; UV (H₂ O) nm (e×10₃)298 (8.1), 236 (15.7), 208 (25.8); IR (KBr) cm⁻¹ 3441, 3275, 1706, 1636,1506, 1176, 1094; MS (FAB⁺) m/z: 419 (MH⁺); HRMS (FAB⁺) calc. for C₁₇H₂₂ F₃ N₄ O₅ 419.1542 (MH⁺), found 419.1558; Anal. calc. for C₁₇ H₂₁ F₃N₄ O₅ : C, 48.81; H, 5.06; N, 13.39, found: C, 48.46; H, 4.98; N, 13.32.

B. Conversion of 13 to5-(6-N-(trifluoroacetyl)-aminohexyl-2'-deoxycytidine (14). ##STR15##

A mixture of 13 (1.379 g, 3.30 mmol) and 10% Pd/C (70 mg) in MeOH (6.6mL) was stirred under H₂ pressure (50 psi). After 20 h the mixture wasfiltered through celite and concentrated. Flash chromatography (SiO₂,MeOH (20%)/CH₂ Cl₂) gave 14: 1.235 g, 89% yield; mp 126-129° C.(MeOH/CH₃ CN); ¹ H NMR (DMSO-d6) δ 1.21-1.33 (m, 4 H), 1.33-1.52 (m, 4H), 1.95 (m, 1 H), 2.06 (ddd, 1 H, J=3.3, 5.9, 13.0 Hz), 2.20 (t, 2 H,J=7.1 Hz), 3.15 (m, 2 H), 3.54 (m, 2 H), 3.74 (m, 1 H), 4.20 (m, 1 H)4.97 (t, 1 H, J=5.0 Hz), 5.16 (d, 1 H, J=4. Hz), 6.15 (t, 1 H, J=6.7Hz), 6.63-6.99 (br s, 1 H), 6.99-7.35 (br s, 1 H), 7.60 (s, 1 H), 9.38(m, 1 H); ¹³ C NMR (DMSO-d6) δ 26.01, 26.59, 27.77, 28.12, 28.24, 39.18,40.32, 61.32, 70.39, 84.77, 87.12, 105.40, 118.28 (CF₃, J_(C-F) =288.4Hz), 138.10, 154.91, 156.11 (COCF₃, J_(C-F) =35.6 Hz), 164.71; UV (H₂ O)nm (e×10³) 280 (7.9); IR (KBr) cm⁻¹ 3423, 3309, 1713, 1668, 1615, 1493,1187, 1155, 1102; MS (FAB⁺) m/z 423 (MH⁺); HRMS (FAB⁺) calc. for C₁₇ H₂₆F₃ N₄ O₅ 423.1855 (MH⁺), found 423.1878; Anal. calc. for C₁₇ H₂₅ F₃ N₄O₅ : C, 48.34; H, 5.97; N, 13.26, found: C, 48.21; H, 5.77; N, 13.19.

C. Conversion of 4 toN4-benzoyl-5-(6-N-(trifluoroacetyl)-aminohexyl)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine(15). ##STR16##

To a mixture of 14 (854 mg, 1.62 mmol) and 4,4'-dimethoxytrityl chloride(585 mg, 1.73 mmol) was added pyridine (15 mL). After stirring for 4 h,80 mL of CH₂ Cl₂ was added, the mixture was extracted with 5% Na₂ CO₃,dried over Na₂ SO₄, and concentrated. Flash chromatography (SiO₂,pyridine (1%)/MeOH (2%)/CH₂ Cl₂) provided 15: 1.130 g, 84% yield: ¹ HNMR (CDCl₃) δ 1.00-1.18 (m, 4 H), 1.2-1.45 (m, 4 H), 1.94 (m, 1 H), 2.23(m, 1 H), 2.34 (m, 1 H), 2.49 (ddd, 1 H, J=3.2, 5.6, 13.4 Hz), 3.16 (m,2 H), 3.32 (dd, 1 H, J=2.3, 10.5 Hz), 3.58 (dd, 1 H, J=2.7, 10.5), 3.80(s, 6 1H), 4.07 (m, 1 H), 4.59 (m, 1 H), 6.25 (m, 1 H), 6.43 (t, 1 H,J=6.6 Hz), 6.85 (d, 4 H, J=7.7 Hz), 7.26-7.38 (m, 6 H), 7.38-7.48 (m, 5H), 7.53 (m, 1 H), 7.76 (s, 1 H), 8.26 (d, 2 H, J=7.3 Hz), 13.1-13.6 (brs, 1 H); ¹³ C NMR (CDCl₃) δ 26.06, 27.37, 28.45, 28.62, 28.77, 39.78,41.23, 55.12, 63.17, 71.89, 85.16, 86.31, 86.60, 113.13, 115.74 (CF₃,J_(C-F) =287.9 Hz), 116.31, 126.98, 127.84, 127.98, 128.05, 129.61,129.95, 132.36, 135.33, 136.94, 144.14, 147.87, 157.05 (COCF₃, J_(C-F)=36.6 Hz), 158.48, 159.32, 179.45; UV (MeOH) nm (e×10³) 332 (21.4), 268(10.0), 236 (25.5); IR (KBr) cm⁻¹ 3331, 1709, 1567, 1509, 1252, 1176; MS(FAB⁺) m/z 829 (MH⁺); HRMS (FAB⁺) calc. for C₄₅ H₄₈ F₃ N₄ O₈ 829.3424(MH⁺), found 829.3436; Anal. calc. for C₄₅ H₄₇ F₃ N4O₈ : C, 65.21; H,5.72; N, 6.76, found: C, 65.57; H, 5.80; N, 6.55.

D. Conversion of 15 to5-(6-N-(trifluoroacetyl)-aminohexyl)-4-N-benzoyl-5'-O-(4,4'-dimethoxy)trityl)-2'-deoxycytidine2-cyanoethyl N,N-diisopropyl phosphoramidite (12). ##STR17##

To a solution of 15 (917 mg, 1.11 mmol) in CH₂ Cl₂ (10 mL) was addeddiisopropylethylamine (0.580 mL, 3.33 mmol) and 2-cyanoethylN,N-diisopropylchlorophosphine (0.275 mL, 1.23 mmol). After 3 h, 50 mLCH₂ Cl₂ was added. The mixture was extracted with saturated NaHCO₃ driedover Na₂ SO₄, and then concentrated. Flash chromatography (SiO₂,pyridine (1%)/MeOH (1%)/CH₂ Cl₂ gave 5: 944 mg, 83% yield; ¹ H NMR(CDCl₃)δ100-1.10 (m, 7 H), 1.10-1.20 (m, 8 H), 1.20-1.40 (m, 5 H),1.80-1.97 (m, 1 H) 2.17-2.30 (m, 1 H), 2.30-2.37 (m, 1 H), 2.39 (m, 1H), 2.50-2.59 (m, 1 H) 2.62 (m, 1 H), 3.13 (m, 2 H), 3.21-3.32 (m, 1 H),3.45-3.68 (m, 4 H), 3.69-3.90 (m, 7 H), 4.12-4.22 (m, 1 H), 4.62-4.73(m, 1 H), 6.15-6.28 (br s, (m, 1 H), 6.43 (m, 1 H), 6.80-6.90 (m, 4 H),7.28-7.38 (m, 6 H), 7.38-7.48 (m, 5 H), 7.48-7.58 (m, 1 H), 7.75-7.83(m, 1 H), 8.23-8.30 (m, 2 H), 13.20-13.50 (br s, 1 H); ¹³ C NMR(CDCl₃)δ20.12, 20.17, 20.37, 20.42, 24.41, 24.49, 24.55, 24.60, 26.12,27.48, 28.55, 28.72, 28.75, 28.86, 39.84, 40.37, 43.10, 43.21, 43.31,55.30, 57.97, 58.12, 58.27, 62.63, 62.79, 73.08, 73.20, 73.43, 73.57,84.96, 85.07, 85.39, 85.43, 85.74, 85.76, 86.67, 113.23, 113.26, 115.82(CF³, J_(C-F) =288.4 Hz), 116.37, 116.39, 117.32, 117.54, 127.13,127.15, 127.96, 128.08, 128.21, 128.27, 129.78, 130.11, 132.39, 135.429136.75, 136.83, 137.23, 144.20, 147.77, 147.80, 155.73, 156.99 (COCF₃,J_(C-F) =37.1 Hz), 158.66, 159.39, 179.55; ³¹ P (CDCl₃) δ 148.62,149.12; MS (FAB⁺) m/z 1029 (MH⁺); HRMS (FAB⁺) calc. for C₅₄ H₆₅ F₃ N₆ O₉P 1029.4503 (MH⁺), found 1029.4551.

Example 4

This example illustrates the synthesis of various dodecanucleotides ofthe present invention.

DNA Synthesis

All oligodeoxynucleotides were synthesized trityl-on using a controlledpore glass solid support via the phosphite-triester method with anApplied Biosystems 391EP DNA synthesizer (1 μmol scale). For thesynthesis of oligomers 16, 17 and 21, controlled pore glass supportderivatized with 7 was prepared by standard methodology. For thesynthesis of oligomer 18, the controlled pore glass support wasderivatized with 11. Cleavage from the solid support and deprotection(except for the 5'-trityl group) was accomplished by treatment withconcentrated NH₄ OH for 15 hours at 55° C. The solution was thenlyophilized with the addition of Et₃ N every hour to inhibitdetritylation, The residue was taken up in 1 mL of 100 mMtriethylammonium acetate (TEAA), pH 7, and purified by reverse phaseHPLC (Hamilton PRP-1, 300 mm×7 mm, Eppendorf CH-30 column heater, 60°C., 23-33% CH₃ CN/100 mM TEAA, pH 7, 20 min., monitored at 260 nm). Thefractions were lyophilized to dryness followed by repeatedlyophilization to dryness with H₂ O (2×1 mL) to remove any residualTEAA. Detritylation was accomplished by treatment with 80% AcOH (0.3 mL)for 20 min. After lyophilization with EtOH (0.3 mL), the residue wastaken up in H₂ O (1 mL), extracted with diethyl ether (3×1 mL) and thenlyophilized to dryness. H₂ O (1 mL) was added to the dry DNA pellet, andthe solution quantified by UV absorbance at 260 nm at 70° C. Theextinction coefficients (at 260 nm) of the natural nucleotides used forcalculations were as follows: dAMP: 15,200; dCMP: 7,700; TMP: 8,830;dGMP 11,500. The extinction coefficients of the unnatural nucleosides at260 nm were determined to be the following:5-(6-aminohexyl)-2'-deoxycytidine: 5,170 (prepared by hydrolysis of 15);5-(6-aminohexyl)-2'-deoxyuridine: 9,200 (prepared by hydrolysis of 10).All oligonucleotide base compositions were confirmed by formic acidhydrolysis followed by HPLC analysis monitored at 260 nm or 270 nm(Alltech, HS, C-18; 20 mM K₂ HPO₄, pH 5.6 (A), MeOH (B), 100% A to 40%B, 20 min.).

Dodecanucleotides 16-21 (Table I) containing 1, 2, and 3 were preparedfrom appropriately protected phosphoramidites using an automated DNAsynthesizer. All oligomers were characterized by digestion toconstituent bases followed by HPLC analysis, 5'-end labeling followed byPAGE and also by laser desorption mass spectrometry. Data for thecorresponding unmodified oligonucleotides 22 and 23 is provided fordirect comparison.

                  TABLE I                                                         ______________________________________                                                      PAGE    Laser Desorptions - MS                                  Oligodeoxynucleotide.sup.a                                                                    Mobility.sup.b                                                                          Cal'd   Found                                       ______________________________________                                        16   5'-d(TTTTTTTTTTTT)                                                                           15.5      3927  3925                                        17 5'-d(TTTTTTTTTTTT) 4.4 4523 4520                                           18 5'-d(TTTTT T T T ) 16.3 3759 3756                                          19 5'-d(CTTTCTCTCCCT) 16.6 3838 3841                                          20 5'-d(CTTTCTCTCCCT) 16.2 3894 3892                                          21 5'-d(CTTTCTCTCCCT) 3.8 4504 4509                                           22 5'-d(CTTTCTCTCCCT) 21.9                                                    23 5'-d(TTTTTTTTTTTT) 20.8                                                  ______________________________________                                         .sup.a T,   and C correspond to zwitterionic monomers 1, 2 and 3              respectively.                                                                 .sup.b Polyacrylamide gel electrophoretic mobility is given in cm from th     origin of the gel.                                                            The samples were electrophoresed in a 20% polyacrylamide, 7M urea             denaturing gel using TrisBorate-EDTA buffer at pH 8.5-9.0.               

Example 5

This example provides the conditions for duplex formation and meltingusing the oligonucleotides 16-23.

UV absorbance versus temperature profiles were measured on an HP 8452Adiode-array UV spectrophotometer in a temperature controlled cell holderwith an HP 89090A peltier temperature controller. The temperature of thecell holder was increased from 0° C. to 90° C. in 1° C. increments at aheating rate of 1°/min. The temperature of the solution was monitored bya thermocouple placed in the cell solution (10 mm path length only) N₂gas (ice-cold) was passed over the cell at low temperatures to avoid thecondensation of moisture. Experiments were performed a minimum of twicewith different samples and T_(m) 's were averaged. Free energy values(in part) and melting temperatures were obtained by non-linearregression using a two-state model An excellent fit of the experimentaldata was seen in all cases. Reverse melting experiments (90° C. to 0°C., 1°/min.) and reduced rate forward melting experiments (0° C. to 90°C., 0.5°/min.) were also performed, and found to give T_(m) 's within0.5° C. Plots of 1/T_(m) versus 1n C_(r) were used to obtainthermodynamic parameters. For the T_(m) versus concentration studies,the following total oligonucleotide concentrations were employed: 140μM, 70 μM, 46 μM, 28 μM, 18 μM, 12 μM, 8.0 μM, 5.0 μM. A 1.0 mm pathlength cell was used for 140 μM to 12 μM concentrations, and a 10 mmpath length cell for 8.0 mM and 5.0 mM concentrations. Chimeric duplexeswere formed between the modified oligonucleotides 16-21 and theircomplementary natural oligonucleotides by mixing 2.5 μM each of 16-21with 2.5 μM of the complementary oligonucleotide (see Table II) in H₂ Oat pH 7 containing 10 m sodium phosphate, 0.1 mM EDTA and the indicatedconcentration of NaCl. Natural duplexes were similarly formed between 22and 23 and their complementary oligomers.

The duplex melting temperatures (under low ionic strength conditions, 50mM NaCl), free energies with complementary natural DNA under low (50 mMNaCl) and high (1M NaCl) ionic strength conditions, and their duplexmelting temperatures with mismatched natural DNA under low ionicstrength conditions (50 mM NaCl) are summarized in Table II.

As Table II indicates, the modified oligonucleotides of the presentinvention, in the presence of complementary DNA, exhibit meltingtemperatures approximately equal to the corresponding natural oligomers22 and 23. Additionally, the presence of positively charged ammoniummoieties and the added steric bulk of the alkyl linking groups had noappreciable effect on the free energy of formation for any of themodified oligonucleotides 16-21. To ensure that all amino groups presentin the duplexes are in their protonated form at pH 7, experiments werealso conducted at pH 5.5 with essentially no change in the results.

                  TABLE II                                                        ______________________________________                                                                 Free Energy of Formation                               T.sub.m C. °) T.sub.m C. °) ΔG ° (Kcal/mol)      Oligo                                                                              T.sub.m C. °                                                                   A-mismatch                                                                              G-mismatch                                                                            50 mM NaCl                                                                            1M NaCl                                ______________________________________                                        16   21.5.sup.a                -3.5                                             17 19.0.sup.a  13.5.sup.c -3.6                                                18 20.0.sup.a   -3.3                                                          19 39.1.sup.b  32.7.sup.d -8.9 -10.3                                          20 45.4.sup.b 26.0.sup.e  -10.6 -12.7                                         21 41.5.sup.b 21.0.sup.e 36.9.sup.d -9.4 -9.4                                 22 39.4.sup.b 20.9.sup.e 32.0.sup.d -9.1 -12.5                                23 22.5.sup.a  11.5.sup.c -4.2                                              ______________________________________                                         .sup.a determined with 5d(A).sub.12                                           .sup.b determined with 5d(AGGGAGAGAAAG)                                       .sup.c determined with 5d(AAAAGAAAAAAA)                                       .sup.d determined with 5d(AGGGAGGGAAAG)                                       .sup.e determined with 5d(AGGGAAAGAAAG)                                  

Example 6

This example illustrates the nuclease resistance of the modifiedoligonuclectides of the present invention.

Into an Eppendorf tube was placed 0.15 optical density units of fullyzwitterionic oligomer 21or natural oligomer 22, 0.03 units of snakevenom phosphodiesterase, 0.48 units of bacterial alkaline phosphatase, 4μL of 100 mM MgCl₂, 8 μL of 0.25 M Tris-HCl (pH 8.1) and 10μL of waterfor a total reaction volume of 40 μL. The mixture was incubated at 37°C. and aliquots were removed at 30 minute time intervals. The amount ofoligomer degradation associated with each aliquot was determined by HPLC(Hamilton PRP-1, 300 mm×7 mm, monitored at 260 nm). Under theseconditions, all of the natural oligomer 22 was consumed after 30minutes, whereas the fully zwitterionic oligomer 21 was not completelydegraded even after 2 hours.

The foregoing is offered primarily for purposes of illustration. It willbe readily apparent to those skilled in the art that the structures,methods, composition components, syntheses and use conditions, and otherparameters of the system described herein may be further modified orsubstituted in various ways without departing from the spirit and scopeof the invention.

What is claimed is:
 1. A composition for binding to an RNA, a DNA, aprotein or a peptide, comprising:a modified oligonuclectides having theformula, ##STR18## wherein n is an integer of from 4 to 30; andeach B isa radical independently selected from the group consisting of, ##STR19##adenine, guanine, thymine and cytosine, wherein each X is a linkinggroup independently selected from the group consisting of C₁ -C₁₀ alkyl,C₁ -C₁₀ unsaturated alkyl, dialkyl ether and dialkylthioether; each Y isa cationic moiety independently selected from the group consisting of--(NH₃)⁺, --(NH₂ R¹)⁺, --(NHR¹ R²)⁺, --(NR¹ R² R³)⁺, dialkylsulfoniumand trialkylphosphonium; and R¹, R², and R³ are each independently loweralkyl having from one to ten carbon atoms, with the proviso that when nis from 4 to 8, no more than 30% Bs are A, G, C or T, and when n is from9 to 30, no more than 50% Bs are A, G, C or T; and an acceptable sterilecarrier, wherein said oligonucleotide is present in an effective bindingamount to an RNA, a DNA a protein, or a peptide.
 2. A method of formingchimeric duplexes between zwitterionic and natural DNA, comprisingtreating natural DNA with a complementary modified oligonuclectideshaving the formula; ##STR20## wherein n is an integer of from 4 to 30;andeach B is a radical independently selected from the group consistingof, ##STR21## adenine, guanine, thymine and cytosine, whereineach X is alinking group independently selected from the group consisting of C₁-C₁₀ alkyl, C₁ -C₁₀ unsaturated alkyl, dialkyl ether anddialkylthioether; each Y is a cationic moiety independently selectedfrom the group consisting of --(NH₃)⁺, --(NH₂ R¹)⁺, --(NHR¹ R²)⁺, --(NR¹R² R³)⁺, dialkylsulfonium and trialkylphosphonium; and R¹, R², and R³are each independently lower alkyl having from one to ten carbon atoms,with the proviso that when n is from 4 to 8, no more than 30% Bs are A,G, C or T, and when n is from 9 to 30, no more than 50% Bs are A, G, Cor T, for a period of time sufficient for duplex formation to occur. 3.A method of claim 2 wherein each B is a radical independently selectedfrom the group consisting of, ##STR22## adenine, guanine, thymine andcytosine, whereineach Y is a cationic moiety independently selected fromthe group consisting of --(NH₃)⁺, --(NH₂ R¹)⁺, --(NHR¹ R²)⁺, and --(NR¹R² R³)⁺.
 4. A method of claim 2, wherein n is an integer of from 4 to15; and each B is a radical independently selected from the groupconsisting of, ##STR23## adenine, guanine, thymine and cytosine,whereineach X is a linking group independently selected from the groupconsisting of C₁ -C₁₀ alkyl and C₁ -C₁₀ unsaturated alkyl; and each Y isa cationic moiety independently selected from the group consisting of--(NH₃)⁺, --(NH₂ R¹)⁺, --(NHR¹ R²)⁺, and (NR¹ R² R³)⁺.
 5. A method ofclaim 2, wherein n is 10 and each B is a radical independently selectedfrom the group consisting of, ##STR24## thymine and cytosine,whereineach X is a linking group independently selected from the groupconsisting of C₃ -C₆ alkyl and C₃ -C₆ unsaturated alkyl; and each Y is--(NH₃)⁺.